3-hydroxypropanal (3HPA) (also called Reuterin and 3-hydroxypropionaldehyde) is a broad spectrum anti-microbial agent produced by a number of microorganisms when fermenting glycerol under anaerobic conditions. Most noted of these are Lactobacillus reuteri (Talarico T. L. et al., Anti Microbial Agents and Chemotherapy (1988), 32, 1854-1858). However, Citrobacter freundii (Daniel, R. et al., J Bacteriol. (1995), 2151-2156), Clostridium butyricum (Malaoui, H. and Marczak, R., Enzyme and Microbial Technology (2000), 27, 399-405), Enterobacter agglomerans (Barbirato, F. et al., Applied and Environ. Microbiol. (1996), 62, 4405-4409) and Klebsiella pneumonia (Sliminiger, P. J. et al., Appl. Environ. Microbiol. (1983), 50, 1444-1450) have all been shown to synthesise 3HPA under similar conditions.
The production of 3HPA is limited by its anti-microbial effect. As levels accumulate in the fermentation broth, it becomes toxic. A maximal level of synthesis of 170 mM of 3HPA has been noted for Lactobacillus reuteri when growing on 200 mM glycerol (Vollenweider, S. et al., J. Agric. Food Chem. (2003), 51, 3287-3293). This currently limits the use of this potentially useful anti-microbial agent to probiotics. Synthesis of this compound at higher levels would be of great commercial advantage.
3HPA is a co-product synthesised by relevant bacteria when metabolising glycerol to 1,3 propandiol via glycerol diol dehydrase, a B12 dependant enzyme (Vollenweider, S. et al., J. Agric. Food Chem. (2003), 51, 3287-3289). 3HPA is normal present as a complex mixture consisting of the 3HPA, its hydrate and its dimer. At higher concentrations (greater than 10%) 3HPA exists predominantly as the dimer. In vivo, the hydrate form is predominant. 3HPA also polymerises to form condensation products and the type of polymer depends on whether growth conditions are acidic or basic.
The anti-microbial effect is thought to be due to activity against sulphydryl containing enzymes, in particular the B1 subunit of ribonucleotide reductase (which accounts for its broad anti-microbial effect) and thioredoxin (Schaefer, L. et al., Microbiology (2010), 156, 1589-1599). 3HPA was found to be active against coliforms at levels of 50-100 U/g (where a unit is equivalent to 5 u/ml) (Daeschel. M. A., Food Technol. (1989), 43, 164-167), against E. coli, P. aeruginosa, S. aureus and B. subtilis at levels of 20-35 ppm (Chen, C. N. et al., J. Biomed. Res. (2002), 61, 360-369) and against protozoa at between 35 and 70 uM (Yunmbam, M. K. and Roberts. J. F., Comp. Biochem. Physiol. C. (1992), 101, 235-238). Producing strains can tolerate up to 30 mM (Barbirato F. et al., Applied Environ. Microbiol. (1996), 62, 1448-1451) and the LD50 in vertebrates (mice) has been recorded as 1,500 U or 7.5 mg (Yunmbam, M. K. and Roberts, J. F. Comp. Biochem. Physiol. C (1993), 105, 521-524).
3HPA is generally produced in a two-step process. In the first step, the bacterium is cultured overnight at 37° C. in MRS broth. The cell mass is then recovered by centrifugation and washed in phosphate buffer. In a second step, the cells are suspended to a dry cell weight of 10 mg/ml in 250 mMo1/1 glycerol and incubated anaerobically at 37° C. Up to 85% of the glycerol is converted to 3HPA. This is a yield of approximately 19 g/l or 1.9%. The key step in this process is the dehydration of glycerol by B12 dependant glycerol dehydratase. The ability to produce 3HPA more readily at higher concentrations, not only as an antibiotic agent but also as a platform molecule for other useful commercial molecules such as 3-hydroxypropionic acid (3HP), which can be converted to esters of 3HP, esters of acrylic acid, acrylic acid (AA), acryamide and acrylic polymers, 1,3 propanediol, and acrolein (Vollenweider, S. and Lacroix, C., Applied Microbiol. (2004), 64, 16-27), would be commercially advantageous. However, 3HPA is generally a complex mixture of molecules whose composition depends on culture conditions such as pH. There have been attempts to commercialize this process via optimisation of glycerol fermentation (Slininger, P. J. and Bothast, R. J., Applied Environ. Microbiol. (1985), 50, 1444-1450) and the trapping of the 3HPA as the semicarbazide to overcome the toxic effects of the compound (Ulmer, C., et al., Chem. Ing. Tech. (2002), 74, 674). Levels of 621 mM of 3HPA have been reported (which is equivalent to 44.71 g per liter) for this method (Talarico, T. L., et al., Antimicrobio. Agents Chemother. (1989), 33, 674-679). However, commercial production through a bacterial system has not yet been successful.
WO2013/011292 describes a microorganism which is capable of producing long chain aliphatic carboxylic acids. This document describes a particular strain referred to as Acetobacter lovaniensis FJ1 having accession number NCIMB 41808 (deposited at NCIMB Ltd. (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA) on 12 Jan. 2011 under the provisions of the Budapest Treaty).